Sanghyeob Lee

The Pepper Transcription Factor CaPF1 Confers Pathogen and Freezing Tolerance in Arabidopsis

An ERF/AP2-type transcription factor (CaPF1) was isolated by differential-display reverse transcription-PCR, following inoculation of the soybean pustule pathogen Xanthomonas axonopodis pv glycines 8ra, which induces hypersensitive response in pepper (Capsicum annuum) leaves. CaPF1 mRNA was induced under conditions of biotic and abiotic stress. Higher levels of CaPF1 transcripts were observed in disease-resistant tissue compared with susceptible tissue. CaPF1 expression was additionally induced using various treatment regimes, including ethephon, methyl jasmonate, and cold stress. To determine the role of CaPF1 in plants, transgenic Arabidopsis and tobacco (Nicotiana tabacum) plants expressing higher levels of CaPF1 were generated. Gene expression analyses of transgenic Arabidopsis and tobacco revealed that the CaPF1 level in transgenic plants affects expression of genes that contain either a GCC or a CRT/DRE box in their promoter regions. Furthermore, transgenic Arabidopsis plants expressing CaPF1 displayed tolerance against freezing temperatures and enhanced resistance to Pseudomonas syringae pv tomato DC3000. Disease tolerance was additionally observed in CaPF1 transgenic tobacco plants. The results collectively indicate that CaPF1 is an ERF/AP2 transcription factor in hot pepper plants that may play dual roles in response to biotic and abiotic stress in plants.

The Tomato Never-ripe Locus Regulates Ethylene-Inducible Gene Expression and Is Linked to a Homolog of the Arabidopsis ETR1 Gene

Fruit ripening represents a complex system of genetic and hormonal regulation of eukaryotic development unique to plants. We are using tomato ripening mutants as tools to elucidate genetic components of ripening regulation and have recently demonstrated that the Never-ripe (Nr) mutant is insensitive to the plant growth regulator ethylene (M.B. Lanahan, H.-C. Yen, J.J. Giovannoni, H.J. Klee [1994] Plant Cell 6: 521–530). We report here ethylene sensitivity over a range of concentrations in normal and Nr tomato seedlings and show that the Nr mutant retains residual sensitivity to as little as 1 part per million of ethylene. Analysis of ripening-related gene expression in normal and mutant ethylene-treated fruit demonstrates that Nr exerts its influence on development at least in part at the level of ethylene-inducible gene expression. We have additionally used cloned tomato and Arabidopsis sequences known to influence ethylene perception as restriction fragment length polymorphism probes, and have identified a tomato locus linked to Nr that hybridizes to the Arabidopsis ETR1 gene at low stringency, suggesting the possibility that Nr may be homologous to ETR1.

Exploitation of pepper EST-SSRs and an SSR-based linkage map.

As genome and cDNA sequencing projects progress, a tremendous amount of sequence information is becoming publicly available. These sequence resources can be exploited for gene discovery and marker development. Simple sequence repeat (SSR) markers are among the most useful because of their great variability, abundance, and ease of analysis. By in silico analysis of 10,232 non-redundant expressed sequence tags (ESTs) in pepper as a source of SSR markers, 1,201 SSRs were found, corresponding to one SSR in every 3.8xa0kb of the ESTs. Eighteen percent of the SSR–ESTs were dinucleotide repeats, 66.0% were trinucleotide, 7.7% tetranucleotide, and 8.2% pentanucleotide; AAG (14%) and AG (12.4%) motifs were the most abundant repeat types. Based on the flanking sequences of these 1,201 SSRs, 812 primer pairs that satisfied melting temperature conditions and PCR product sizes were designed. 513 SSRs (63.1%) were successfully amplified and 150 of them (29.2%) showed polymorphism between Capsicum annuum ‘TF68’ and C. chinense ‘Habanero’. Dinucleotide SSRs and EST–SSR markers containing AC-motifs were the most polymorphic. Polymorphism increased with repeat length and repeat number. The polymorphic EST–SSRs were mapped onto the previously generated pepper linkage map, using 107 F2 individuals from an interspecific cross of TF68xa0xa0×xa0xa0Habanero. One-hundred and thirtynine EST–SSRs were located on the linkage map in addition to 41 previous SSRs and 63 RFLP markers, forming 14 linkage groups (LGs) and spanning 2,201.5xa0cM. The EST–SSR markers were distributed over all the LGs. This SSR-based map will be useful as a reference map in Capsicum and should facilitate the use of molecular markers in pepper breeding.

EST and microarray analyses of pathogen-responsive genes in hot pepper (Capsicum annuum L.) non-host resistance against soybean pustule pathogen (Xanthomonas axonopodis pv. glycines).

AbstractLarge-scale single-pass sequencing of cDNA libraries and microarray analysis have proven to be useful tools for discovering new genes and studying gene expression. As a first step in elucidating the defense mechanisms in hot pepper plants, a total of 8,525 expressed sequence tags (ESTs) were generated and analyzed in silico. The cDNA microarray analysis identified 613 hot pepper genes that were transcriptionally responsive to the non-host soybean pustule pathogen Xanthomonas axonopodis pv. glycinesn(Xag). Several functional types of genes, including those involved in cell wall modification/biosynthesis, transport, signaling pathways and divergent defense reactions, were induced at the early stage of Xag infiltration. In contrast, genes encoding proteins that are involved in photosynthesis, carbohydrate metabolism and the synthesis of chloroplast biogenetic proteins were down-regulated at the late stage of Xagninfiltration. These expression profiles share common features with the expression profiles elicited by other stresses, such as fungal challenge, wounding, cold, drought and high salinity. However, we also identified several novel transcription factors that may be specifically involved in the defense reaction of the hot pepper. We also found that the defense reaction of the hot pepper may involve the deactivation of gibberellin. Furthermore, many genes encoding proteins with unknown function were identified. Functional analysis of these genes may broaden our understanding of non-host resistance. This study is the first report of large-scale sequencing and non-host defense transcriptome analysis of the hot pepper plant species. (The sequence data in this paper have been submitted to the dbEST and GenBank database under the codes 10227604–10236595 and BM059564–BM068555, respectively. Additional information is available at http://plant.pdrc.re.kr/ks200201/pepper.html).

Expression of a novel NAC domain-containing transcription factor (CaNAC1) is preferentially associated with incompatible interactions between chili pepper and pathogens

We aim to isolate genes in chili pepper that are regulated during the hypersensitive response to infection by nonhost pathogens, with a view to elucidating the defense responses against pathogen attack. Among the 90 transcription factors initially characterized by reverse RNA gel blot analysis, a cDNA clone, CaNAC1 (Capsicum annuum NAC1) containing the plant-specific NAC domain motif was further characterized. Expression of the CaNAC1 gene was rapidly and specifically induced during incompatible interactions between pepper and bacterial or viral pathogens. Additionally, this gene was strongly induced by exogenously applied salicylic acid and ethephon, whereas methyl jasmonate only had a transient effect. A CaNAC1-smGFP (soluble modified green fluorescent protein) fusion protein localized to the nucleus following transfection into the epidermis of onion. Using the yeast system, we further disclose that the transcription activation domain of CaNAC1 is located in the C-terminal half of the protein. Our results collectively suggest that the plant-specific NAC domain protein, CaNAC1, may play a role in the regulation of defense responses in plants.

Non-climacteric fruit ripening in pepper: increased transcription of EIL- like genes normally regulated by ethylene

Only limited information has been published to date on the similarities and differences between climacteric and non-climacteric fruit ripening on transcriptional level. To address this issue, we performed a direct comparative transcriptome analysis between tomato and pepper fruits using heterologous microarray hybridization. Given the significant differences in the morphological, physiological, and biochemical characteristics of pepper and tomato fruits, the existence of extensive common regulons is surprising. This finding suggests the conservation of ripening mechanisms in climacteric and non-climacteric fruits. However, disparate expression profiles were also observed in both fruits. This study revealed that a gene that encodes an enzyme that converts lycopene to downstream carotenoids is induced in pepper but not in tomato. Most of the genes that encode ribosomal proteins are only induced in early fruit-stage pepper fruit and show rapidly diminishing expression in the later developmental stages. The genes involved in ethylene biosynthesis were not induced in pepper fruit. However, the EIL-like genes, ethylene-mediated signaling components, were induced in pepper fruit. Divergent types of transcription factors were expressed in ripening tomato and pepper fruits, suggesting they may be key factors that differentiate these distinct ripening processes.

Molecular and biochemical characterization of the Capsicum annuum calcium-dependent protein kinase 3 (CaCDPK3) gene induced by abiotic and biotic stresses.

The isolated full-length Capsicum annuum calcium-dependent protein kinasexa03 (CaCDPK3) cDNA clone was selected from the chili pepper expressed sequence tag database (http://www.pdrc.re.kr/ks200201/pepper.html). Phylogenetic analysis based on the deduced amino acid sequence of CaCDPK3 cDNA revealed significant sequence similarity to the winter squash (Cucurbita maxima) CmCPK2 gene (81% identity). Genomic gel blot analysis disclosed that CaCDPK3 belongs to a multigene family in the pepper genome. CaCDPK3 expression was root tissue-specific, as shown by Northern blot data. The gene was rapidly induced in response to various osmotic stress factors and exogenous abscisic acid application in pepper leaves. Moreover, CaCDPK3 RNA expression was induced by an incompatible pathogen and by plant defense-related chemicals such as ethephon, salicylic acid and jasmonic acid. The biochemical properties of CaCDPK3 were investigated using a CaCDPK3 and glutathione S-transferase (GST) fusion protein. The recombinant proteins retained calcium-binding ability, and displayed autophosphorylation activity in vitro in a calcium-dependent manner. Further transient-expression studies showed that CaCDPK3 fused with soluble modified green fluorescent protein (smGFP) localized to the cytosol in chili pepper protoplasts. We propose that CaCDPK3 is implicated in biotic and abiotic stresses in pepper plants.

Integration of Cytogenetic and Genetic Linkage Maps Unveils the Physical Architecture of Tomato Chromosome 2

We report the integration of the linkage map of tomato chromosome 2 with a high-density bacterial artificial chromosome fluorescence in situ hybridization (BAC–FISH)-based cytogenetic map. The euchromatic block of chromosome 2 resides between 13 and 142 cM and has a physical length of 48.12 μm, with 1 μm equivalent to 540 kb. BAC–FISH resolved a pair of loci that were 3.7–3.9 Mb apart and were not resolved on the linkage map. Most of the regions had crossover densities close to the mean of ∼200 kb/cM. Relatively hot and cold spots of recombination were unevenly distributed along the chromosome. The distribution of centimorgan/micrometer values was similar to the previously reported recombination nodule distribution along the pachytene chromosome. FISH-based physical maps will play an important role in advanced genomics research for tomato, including map-based cloning of agronomically important traits and whole-genome sequencing.

Insight into Types I and II nonhost resistance using expression patterns of defense-related genes in tobacco

Plants protect themselves against pathogens using a range of response mechanisms. There are two categories of nonhost resistance: Type I, which does not result in visible cell death; and Type II, which entails localized programmed cell death (or hypersensitive response) in response to nonhost pathogens. The genes responsible for these two systems have not yet been intensively investigated at the molecular level. Using tobacco plants (Nicotiana tabacum), we compared expression of 12 defense-related genes between a Type I (Xanthomonas axonopodis pv. glycines 8ra) nonhost interaction, and two Type II (Pseudomonas syringae pv. syringae 61 and P. syringae pv. phaseolicola NPS3121) nonhost interactions, as well as those expressed during R gene-mediated resistance to Tobacco mosaic virus. In general, expression of most defense-related genes during R gene-mediated resistance was activated 48xa0h after challenge by TMV; the same genes were upregulated as early as 9xa0h after infiltration by nonhost pathogens. Surprisingly, X. axonopodis pv. glycines (Type I) elicited the same set of defense-related genes as did two pathovars of P. syringae, despite the absence of visible cell death. In two examples of Type II nonhost interactions, P. syringae pv. phaseolicola NPS3121 produced an expression profile more closely resembling that of X. axonopodis pv. glycines 8ra, than that of P. syringae pv. syringae 61. These results suggest that Type I nonhost resistance may act as a mechanism providing a more specific and active defense response against a broad range of potential pathogens.

Transcriptional Regulation of the Respiratory Genes in the Cyanobacterium Synechocystis sp. PCC 6803 during the Early Response to Glucose Feeding

The coordinated expression of the genes involved in respiration in the photosynthetic cyanobacterium Synechocystis sp. PCC 6803 during the early period of glucose (Glc) treatment is poorly understood. When photoautotrophically grown cells were supplemented with 10 mm Glc in the light or after a dark adaptation period of 14 h, significant increases in the respiratory activity, as determined by NAD(P)H turnover, respiratory O2 uptake rate, and cytosolic alkalization, were observed. At the same time, the transcript levels of 18 genes coding for enzymes associated with respiration increased with differential induction kinetics; these genes were classified into three groups based on their half-rising times. Transcript levels of the four genes gpi, zwf, pdhB, and atpB started to increase along with a net increase in NAD(P)H, while the onset of net NAD(P)H consumption coincided with an increase in those of the genes tktA, ppc, pdhD, icd, ndhD2, ndbA, ctaD1, cydA, and atpE. In contrast, the expression of the atpI/G/D/A/C genes coding for ATP synthase subunits was the slowest among respiratory genes and their expression started to accumulate only after the establishment of cytosolic alkalization. These differential effects of Glc on the transcript levels of respiratory genes were not observed by inactivation of the genes encoding the Glc transporter or glucokinase. In addition, several Glc analogs could not mimic the effects of Glc. Our findings suggest that genes encoding some enzymes involved in central carbon metabolism and oxidative phosphorylation are coordinately regulated at the transcriptional level during the switch of nutritional mode.